A rapid freeze-quench setup for multi-frequency EPR spectroscopy of enzymatic reactions

Roberta Pievo, Brigitta Angerstein, Alistair J. Fielding, Christian Koch, Ivo Feussner, Marina Bennati

    Research output: Contribution to journalArticlepeer-review

    Abstract

    Electron paramagnetic resonance (EPR) spectroscopy in combination with the rapid freeze-quench (RFQ) technique is a well-established method to trap and characterize intermediates in chemical or enzymatic reactions at the millisecond or even shorter time scales. The method is particularly powerful for mechanistic studies of enzymatic reactions when combined with high-frequency EPR (ν≥90 GHz), which permits the identification of substrate or protein radical intermediates by their electronic g values. In this work, we describe a new custom-designed micro-mix rapid freeze-quench apparatus, for which reagent volumes for biological samples as small as 20 μL are required. The apparatus was implemented with homemade sample collectors appropriate for 9, 34, and 94 GHz EPR capillaries (4, 2, and 0.87 mm outer diameter, respectively) and the performance was evaluated. We demonstrate the application potential of the RFQ apparatus by following the enzymatic reaction of PpoA, a fungal dioxygenase producing hydro(pero)xylated fatty acids. The larger spectral resolution at 94 GHz allows the discernment of structural changes in the EPR spectra, which are not detectable in the same samples at the standard 9 GHz frequency. A powerful combination! The performance of a new micro rapid freeze-quench apparatus combined with electron paramagnetic resonance (EPR) spectroscopy is reported, and the ease of setting up a multi-frequency EPR analysis to investigate the kinetics and mechanisms of enzymatic processes is demonstrated. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    Original languageEnglish
    Pages (from-to)4094-4101
    Number of pages7
    JournalChemPhysChem
    Volume14
    Issue number18
    DOIs
    Publication statusPublished - 16 Dec 2013

    Keywords

    • enzymatic reactions
    • EPR spectroscopy
    • radical intermediates
    • rapid freeze quench (RFQ)
    • tyrosyl

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